Live rabies virus vaccine and method for the production thereof



United States Patent 3 255 080 LIVE RABIES VIRUS VAClNE AND METHGD FORTHE PRODUCTION rrrnnuor Jerrell B. Emery, Zionsville, Ind, assignor toThe Dow,

This invention is concerned with the preparation of livemodified rabiesvirus and is particularly directed to a novel method for propagatinglive modified rabies virus by a tissue culture technique and to animproved vaccine produced by such method.

The classical method for preparing rabies vaccine involves propagationof the raibies virus in living mammals such as rabbits. After the virusis fixed by serial passage through rabbits, for example, infectednervous tissue, such as the brain or spinal cord, from such rabbits isattenuated by drying or chemical treatment and suspensions of suchtissue of graded potency are injected to immunize the recipient againstrabies. The injection of such nervous tissue in some cases produceshighly undesirable side effects.

Rabies virus has previously been cultured outside living animalsprimarily in suspensions of minced nervous tissue as, for example, inmouse embryo brain or rabbit embryo brain. Prior workers with rabiesvirus in tissue culture have also generally found it necessary to addserum, plasma or the like proteinaceous fluids to their culture media inorder to obtain reproduction of the virus. Plotz and Reagen (Science 95,102404 (1942)) have reported, for example, the culturing of a streetvirus strain of rabies employing chick embryo cells grown by the plasmaclot method. The latter authors have indicated that the use ofproliferating cells, such as are obtained in the plasma clot technique,is a requirement for obtaining rapid reproduction of the virus. I

Another approach to the reproduction of rabies virus has been byadaptation of the virus to growth in embryonated eggs. The so-calledFlury strain of rabies virus has found wide use in preparing vaccinesfor the immunization of dogs against rabies whereby said vaccine isgrown in embryonated eggs. The Flury strain was isolated by passage ofan original street virus through chick brains, and after serialintracerebral passage in baby chicks, was adapted to growth inembryonated eggs. The chick-embryo-adapted virus may be injectedintramuscularly in dogs without producing rabies symptoms and yetfosters the production of rabies antibodies in the injected animal.Although such production of the Flury strain of rabies vaccine hasadvantages over older methods, it is handicapped by the retention of ahigh proportion of chicken protein in the finished vaccine and by thefact that it is ditficult to remove contaminants such as other virusesor pleuro-pneumonia type organisms form virus propagated by egg passage.This process is further handicapped by the inherent expense involved inthe production of vaccine in embryonated eggs.

It is an object of the present invention to provide a novel and improvedrabies vaccine. It is a further object of the invention to provide amethod for culturing modified live rabies virus in chick embryo tissueculture. A particular object is to provide a method for culturing suchrabies virus in chick embryo tissue culture employing a culture mediumfree of serum or other proteinaceous additaments. Yet another object isto provide a rabies vaccine substantially free of undesirablecontaminating viruses. Other objects will become apparent from thefollowing specification and claims. I

In accordance with the present invention, it has been az ssnas PatentedJune 7, 1966 hce discovered that modified live rabies virus,particularly the Flury strain of said virus, may be propagated in chickembryo tissue culture employing a synthetic maintenance medium withoutserum or other proteinaceous additament. It has further been discoveredthat the application of successive terminal dilution cultures in suchtissue culture method produces a virus product substantially free ofcontaminating viruses and other organisms. It is among the advantages ofthe invention that vaccines produced in accordance therewith aresubstantially free of chick protein and have high antigenicity forproducing immunization against rabies without undesirable side effects.

In carrying out the invention, eg -adapted, live modified rabies virusof the Flury strain is propagated in tissue cultures of embryonic chickcells. Said virus of the Flury strain has been adapted to be propagatedin embryonated eggs after having been modified from the original streetstrain by 138 successive pass-ages by intracerebral inoculation inchicks. In the preparation of tissue cultures, 7- to IO-day-old chic-kembryos are minced and dispersed in any suitable fashion to obtain achick embryo cell dispersion. Portions of such dispersion are placed inculture vessels with a nutrient medium capable of supporting growth andmultiplication of the cells and maintained under good growth conditionsfor such cells. As soon as good monolayer growth of chick embryo tissuecells is obtained, the nutrient medium is poured off and an inoculum ofthe aforementioned egg-adapted virus introduced into the tissue culture.A synthetic maintenance medium is added to each culture vessel, eitherbefore or after introducing the inoculum, in amount to cover the celllayers. The vessels are then maintained at a temperature of about 34-37C. for a period of time, usually from about 4 to 14 days, to accomplishmultiplication of the virus.

The multiplication of rabies virus in chick embryo tissue culture is notevidenced by-any observable cytopathological effect on the tissue cells.Growth of the virus is demonstrated by intracerebral inoculation intomice of serial dilutions of the fluid from the culture tubes followingthe above-described incubation of the virus.

The preferred mode of propagation and purification of the rabies virusfor the preparation of vaccines includes one or more limiting dilutionpassages. In such technique, after it has been established by ti-teringin mice that multiplication of the virus in tissue culture isproceeding, virus is harvested by freezing and thawing the incubatedculture medium and cells about 5 to 14 days after inoculation with thevirus, followed by separation of a clear, supernatant suspension of thevirus freed of cell debris. The resulting virus suspension is diluted insuccessive 10-fold steps and a portion of each such dilution inoculatedinto chick embryo tissue culture in the abovedescribed fashion.Thereafter, the inoculated culture tubes are maintained under conditionsfavorable to growth of the virus and tested periodically byintracerebral injection in mice to ascertain the presence or absence ofrabies virus. Virus harvested from the culture prepared from the mostdilute inoculum showing growth of the virus is then employed as theinoculum for further serial passages of the virus. In general, it isdesirable to repeat serially such limiting dilution passages at leastabout 3 times and preferably, at least about 10 times, to assure thedesired purification of the virus. Thereafter, the purified virus fromthe final limiting dilution passage may be multiplied by one or morefurther tissue culture passage to produce any desired quantity ofvaccine.

In the preferred mode of operation, the chick embryo tissue cultures areprepared by rnincing chick embryos after removing the eyes, and thendispersing the cells thereof by trypsinization. Portions of theresulting cell suspension are placed in culture vessels with a suitablenutrient medium, such as a solution of lactalbumin hydrolysate in Earlesbasic salt solution fortified with an animal serum such as inactivatedcalf serum and containing suitable antibiotics to suppress bacterialcontamination. When good m-onolayer .growth of chick embryo tissue cellshas been so obtained, the nutrient medium is decanted and the cell sheetof the tissue culture is wetted with an inoculum of the above-describedegg-adapted Flury strain of rabies virus. In such operations, it appearscritical for obtaining good yields of propagated virus to control theconcentration of virus and/ or egg material in the inoculum. Goodresults have been obtained when employing an inoculum consisting of asuspension of about 1 percent of embryo material from an active cultureof the Flury strain of rabies virus in embryonated eggs.

In this preferred mode of operation, following the wet ting of the chickembryo cell sheet with the suspension of active virus, the culturevessels are closed and maintained stationary for a period of time topromote absorption of the virus by the cells. Thereafter, a syntheticmaintenance medium, such as medium 199 of Morgan, Morton and Parker, orEagles basal medium, is added to the inoculated culture vessel and suchvessels are maintained at a temperature of about 35 for a period ofabout 4 to 14 days, preferably about 7 to 9 days, to accomplishmultiplication of the virus. Subsequent passages of the resultingpropagated virus may be carried out by the herein-described adsorptiontechnique or by introducing a small quantity of harvested virus from aprevious tissue culture passage into the culture tube along with orsubsequent to the introduction of the synthetic maintenance medium. Theterm synthetic maintenance medium as employed herein refers to a mediumcomposed of essential amino acids, purines, sugars, vitamins andinorganic salts capable of maintaining cultured cells in condition formultiplication of virus without supplements such as serum obtained fromliving animals.

For the preparation of vaccines, the virus produced by serial passage inchick embyro tissue culture, as described herein, may be harvested inany suitable fashion. In the preferred method of operation, themaintenance medium, containing a portion of the virus produced, isdecanted from the tissue culture about 7 days after the inoculationof-the tissue culture with the virus. Following the decantation, anequal volume of a suitable vaccine stabilizer, such as a caseinhydrolysate, an amino acid-sugar mixture, a buffered sugar solution orthe like, is introduced into the culture vessel and the contents thereofare then frozen and thawed to rupture the tissue cells and release thevirus therefrom. The stabilizer fluid is then decanted, pooled with theprior harvest of mantenance medium and centrifuged to remove cell debrisand produce a vaccine product. The latter can then be titered forpotency and tested for purity in conventional fashion. If desired, suchvaccine may be stored directly at low temperature or may be preserved insealed vessels after freeze-drying.

In a representative operation, illustrative but not limiting of theinvention, live rabies virus of the Flury strain,

which is characterized as having been modified from the originallyisolated street virus by 138 successive passages by intracerebralinoculation in chicks followed by 64 passages in embryonated eggs, wasinoculated into tissue cultures of chick embyro cells. The tissuecultures were monolayer cultures grown in glass culture vessels. Theculture material was prepared as follows.

Sevento ten-day-old chick embryos were finely chopped under asepticconditions, after removal of the eyes, and the resulting minced tissuewashed with phosphite-buffered saline solution. The washed tissue wastrypsinized in conventional fashion with agitation by a magneticstirrer. The trypsinized tissue was centrifuged at 1000 r.p.m. for 3minutes and the resulting concentrated cells were suspended in nutrientmedium to provide about 500,000 to.600,000 cells per milliliter. Thenutrient 4 medium was prepared with 'Earles basic salt solutionconsisting of sodium chloride 6.8 g., potassium chloride 0.4 g., calciumchloride 0.2 g., magnesium sulfate heptahydrate 0.2 g., monosodium acidphosphate monohydrate 0.125 g., glucose 1.0 g., sodium bicarbonate 2.2g., and phenol red 0.02 g. dissolved in distilled water to make 1000milliliters of finished solution. Said nutrient medium had the followingcomposition:

Ingredient: 1 Percent Earles basic salt solution (EBSS) 85 5% solutionof lactalbumin hydrolysate in EBSS without sodium bicarbonate 10Inactivated calf serum 5 The nutrient medium further contained 200 unitsof penicillin and 200 micrograms of streptomycin per milliliter.

One-milliliter portions of the above chick embryo cell suspension wereplaced in sterile tubes and maintained at a temperature of about 35 C.for 24 to 48 hours until a good growth of tissue was obtained. A similarprocedure was also used to produce larger batches by incubating tomilliliters of the cell suspension in Blake or Roux culture flasks.Following the initial incubation period for the chick embryo tissuecells, the nutrient medium was drained from the cell sheets and thelatter washed with a synthetic maintenance medium, namely medium 199 ofMorgan et al. (Proc. Soc. Experimental Biology and Medicine, 1950, 73,pp. 1-8), supplemented with 200 units of penicillin and 200 microgramsof streptomycin per milliliter. The wash medium was drained from thecell sheets of cultured tissue, and 0.2 milliliter of an aqueoussuspension of 1 percent of embryo material from embryonated eggs, inwhich the Flury strain of rabies virus had been propagated, was addedand distributed so as to contact the cell sheet. This egg virus materialhad a titer of 10 mouse lethal doses per 0.2 milliliter as determined byintracerebral inoculation in mice. The culture tube and contents sotreated were maintained at 35 C. for one hour, with the tubes held in astationary position such that the inoculum covered the cell sheet. Oncompletion of this adsorption period, 2 milliliters of medium 199 wasadded to the culture and the tube and contents placed in a roller drumand agitated slowly by rolling for seven days at .a temperature of 35 C.

On the seventh day after the above inoculation of the tissue culturewith the egg virus, the culture medium was frozen and thawed to releaseintracellular virus, and the resulting mixture was centrifuged to throwdown cell debri-s. Two-tenths milliliter of vthesupernatant liquid fromthe centrifuged mixture was employed as the inoculum in fresh tubes ofcultured tissue'as before. After adsorption of the virus in theabove-described manner, incubation was carried out for seven days at 35,as previously. After siX such passages, subsequent passages were made byinoculating 0.2 milliliter of supernatant virus-containmg fluid from theimmediately preceding passages into a fresh, washed, tissue culturecontaining 2 milliliters of maintenance medium. After 4 such additionalpassages, the supernatant liquid from the culture, after freezing andthawing, was titered by intracerebral inoculation in mice .and found tohave a titer of 10 per 0.03 milliliter.

This passage level represented a dilution factor of 10 from the originalstarting virus.

I Commencing at the 14th passage level, a series of blind,limiting-dilution, purification passages were made in the followingmanner. Fourteenth passage level virus was diluted in IO-fold steps andinoculated in 0.2 milliliter amounts into chick embryo cultures. At 7days, the cultures inoculated with 10- and 10* dilutions were harvestedby freezing and thawing the cell sheet in order to rupture the cells. At10 days after inoculation, the cultures inoculated with 10- and 10"dilutions were harvested in a similar manner. All harvests were testedin mice for rabies virus. The cultures showing growth ofmouse-infectious virus from the most dilute inoculum were used as seedwhich was diluted serially and passed into chick embryo tissue culturesas before. After eight such limiting-dilution purification passages,which represented a dilution factor of l() from the original embryonatedegg virus inoculum, the virus harvested from the eighthlimiting-dilution passage titered lO per 0.03 milliliter, or a increasein virus over the starting material. Ten such limiting-dilution passageswere made to assure freedom of the final product from contaminatingviruses and other microogranisms.

Bottles prepared from trypsinized chick embryo tissue are used forlarger quantities of virus. Bottles were inoculated by adsorbing amixture of 1.0 milliliter of virus suspension from a previous pasage and9 milliliters of medium 199 to the cell sheet for one hour at 35 C. Then90 milliliters of medium 199 were added'and the bottles incubatedstationary. Ten days after inoculation, the infectious supernatant fiuidwas removed and an equal quantity of a suitable stabilizer was added tothe cell sheet which was then ruptured by freezing and thawing. Celldebris and stabilizer were combined with the supernatant fluid andcentrifuged to remove cell debris to prepare a finished vaccine.

Other synthetic maintenance media, such as Parker and Healys syntheticmedium No. 858 or Eagles medium, can be employed in the above procedureinstead of medium 199, if desired.

Virus harvested from the 4th, 5th, 6th and 7th tissue culture passages,as described above, was pooled and employed as a vaccine forintramuscular injection in dogs. This pooled vaccine was found to have atiter of 10 mouse lethal doses 50 percent (MLD per milliliter. A dose ofone milliliter of this vaccine was injected into each of 5 dogs and 6similar dogs were maintained as uninoculated controls. About 8 weeksafter the inoculation, all of the dogs were challenged by injection of asuspension of virulent rabies virus. All of the inoculated dogs survivedin good condition while all of the uninoculated control dogs died.

T 0 demonstrate the improved potency of vaccines prepared in accordanceWith the invention, particularly after purification by theabove-described limiting-dilution technique, guinea pigs were inoculatedintramuscularly with vaccines as follows:

(1) Standard commercial Flury strain vaccine from embryonated eggculture.

(2) Virus suspension from chick embryo tissue culture (TC) beforelimiting-dilution purification.

(3) Virus suspension from chick embryo tissue culture after 10limiting-dilution passages.

A portion of each vaccine was diluted serially to give dilutions of 1010- 10* and 10 respectively, based on the undiluted vaccine. A group ofguinea pigs was inoculated with the undiluted vaccine in each instanceand further groups with each of the serial dilutions of theabove-described vaccines. Each guinea pig received an injection of 0.25milliliter of one of the vaccines or dilutions thereof intramuscularly.Other groups of similar guinea pigs were maintained uninoculated toserve as controls. 7

Twenty-one days after the inoculation, all the guinea pigs werechallenged with a virulent street rabies virus by intramuscularinjection of an amount of said virulent virus standardized to cause atleast 80 percent mortality in unvaccinated guinea pigs. The guinea pigswere then held and observed for signs of paralysis. From the number ofguinea pigs protected with each dilution of vaccine employed, the 50percent protective end point for each vaccine was calculated by standardmethods. The results are summarized in the following table.

Mouse doses MLD) Vaccine Number Source Required to Protect 50% oi,Guinea Pigs Embryonated egg 242 Chick embryo TC, Unpurified 184 Chickembryo TC, Purified 35 Eighty-seven percent of the unvaccinated controlswere paralyzed by the rabies challenge virus. I

The foregoing results demonstrate the improved antigenicity of therabies virus cultured on chick embryo tissues. This improvedantigenicity was obtained with no increase in virulence of the virus fordogs or guinea pigs.

I claim:

1. A method for propagating a modified live rabies virus which comprisesthe steps of culturing chick embryo cells in vitro in a medium capableof fostering growth of such cells to produce monolayer tissue cultures,decanting the growth medium from the cultured cells, washing said cellswith a protein-free nutrient medium, inoculating the cultured cells withan aqueous suspension of live rabies virus of the Flury strain which hasbeen further modified by at least about 64 successive passages inembryonated eggs, adding to the cultured cells a synthetic maintenancemedium selected from the group consisting of medium 199, medium 858 andEagles medium and maintaining the resulting culture at a temperature offrom about 34 C. to 37 C. for a period of from about 4 to 14 days toaccomplish multiplication of the virus in said cells.

2. A method according to claim 1 in which the inoculated culture ismaintained at a temperature of about 35 C. for a period of seven daysand thereafter, the modified live virus is harvested by decanting themaintenance medium, adding to the cell sheet a stabilizer selected fromthe group consisting of a solution of casein hydrolysate, a solution ofan amino acid-sugar mixture and a buffered sugar solution, freezing andthawing the resulting mixture to rupture the cells and release virustherefrom, combining the resulting virus suspension in stabilizer withthe decanted maintenance medium and separating the combined virussuspension from the cells and cell debris by centrifugation.

3. The method of claim 2 wherein the harvested virus is freeze-dried toproduce a vaccine concentrate.

4. A method for propagating a modified live rabies virus which comprisesthe steps of culturing chick embryo cells in vitro in a medium capableof fostering growth of such cells to produce monolayer tissue cultures,decanting the growth medium from the cultured cell sheet, washing saidcells with a protein-free nutrient medium, wetting said cell sheet witha minimal quantity of an aqueous suspension of the modified live rabiesvirus, maintaining said suspension in contact with the cell sheet for aperiod of time at a temperature of from about 34 C. to about 37 C. toaccomplish adsorption of the virus by the cells, adding a maintenancemedium selected from the group consisting of medium 199, medium 858 andEagles medium to cover the cell sheet and maintaining the culture at atemperature of about 35 C. for a period of from about 4 to 14 days toaccomplish multi- 0 plication of the virus in said cells.

5. A method according to claim 4 wherein the inoculated culture withmaintenance medium is held at a temperature of about 34 C. to 37 C. fora period of from about 4 to 14 days and thereafter, a suspension of thevirus is harvested from the culture by decanting the maintenance medium,adding to the cell sheet a stabilizer selected from the group consistingof a solution of casein hydrolysate, a solution of an amino acid-sugarmixture and a buffered sugar solution, freezing and thawing theresulting mixture to rupture the cells and release virus therefrom,combining the resulting virus suspension in stabilizer with the decantedmaintenance medium and separating the combined virus suspension from thecells and cell debris by centrifugation.

, 6. A method according to claim 4 wherein the inoculated culture withmaintenance medium is held at a temperature of about 35 C. for a periodof from about 7 to 9 days and thereafter, a suspension of the virus isharvested from the culture by decanting the maintenance medium, addingto the cell sheet a stabilizer selected from the group consisting of asolution of casein hydrolysate, a solution of an amino acid-sugarmixture and a buffered sugar solution, freezing and thawing theresulting mixture to rupture the cells and release virus therefrom,combining the resulting virus suspension'in stabilizer with the decantedmaintenance" medium and separating the combined virus suspension fromthe cells and cell debris by centrifugation.

7. A method for preparing a modified live rabies virus vaccine whichcomprises the steps of inoculating the Flury strain of rabies virusfurther modified by at least about 64 successive passages in embryonatedeggs into a culture of chick embryo cells, maintaining the inoculatedculture for a period of time to accomplish multiplication of the virus,harvesting virus from such culture, preparing serial, successive,10-fold dilutions of the harvested virus, inoculating each such dilutioninto a fresh chick embryo tissue culture, harvesting virus from theculture prepared from the most dilute inoculum from which reproductionof the virus can be demonstrated by intracerebral inoculation in mice,employing the resulting harvested virus as seed for the next series ofdilution passages and repeating such limiting dilution tissue culturepassages for a total of at least about 10 such passages, maintaining thefinal tissue culture from the most dilute inoculum from whichreproduction of the virus can be demonstrated until substantialmultiplication of the virus therein is obtained and harvesting therefroma modified live rabies virus substantially free of contaminating virusesand other organisms.

8. A method according to claim 7 wherein the virus harvested from thefinal limiting dilution passage is multiplied in at least one furtherchick embryo tissue culture passage wherein an established monolayertissue culture of chick embryo cells is washed with a proteinfreenutrient medium, the resulting washed cell sheet is inoculated with saidvirus, a synthetic maintenance medium selected from the group consistingof medium 199, medium 858 and Eagles medium is added thereto, theresulting culture is maintained at a temperature of from about 34 to 37C. for a period of from about 4 to 14 days and the propagated virusharvested by decanting the maintenance medium, adding to the cell sheeta stabilizer selected from the group consisting of a solution of caseinhydrolysate, a solution of an amino acid-sugar mixture and a bufferedsugar solution, freezing and thawin-g the resulting mixture to rupturethe cells and release virus therefrom, combining the resulting virussuspension in stabilizer with the decanted maintenance medium andseparating the combined virus suspension from the cells and cell debrisby centrifugation to produce a proteinaceous additaments and ofcontaminating viruses prepared by the method of claim 7,

10. A modified live rabies vaccine prepared by the steps of culturingchick embryo cells in vitro in a medium capable of fostering growth ofsuch cells to produce monolayer tissue cultures, decanting the growthmedium from the cultured cells, washing said cells with a protein-freemaintenance medium, inoculating the cultured cells with an aqueoussuspension of live rabies virus of the Flury strain, further modified byat least about 64 successive passages in embryonated eggs, adding to thecultured cells a synthetic maintenance medium selected from the groupconsisting of medium 199, medium 858 and Eagles medium, maintaining theresulting culture at a temperature of from about 34 C. to 37 C. for aperiod of time to accomplish multiplication of the virus in said cellsand harvesting the virus by decanting the maintenance medium, adding tothe cell sheet a stabilizer selected from the group consisting of asolution of casein hydrolysate, a solution of an amino acid-sugarmixture and a buttered sugar solution, freezing and thawing theresulting mixture to rupture the cells and release virus therefrom,combining the resulting virus suspension in stabilizer with the decantedmaintenance medium and separating the combined virus suspension from thecells and cell debris by centrifugation.

References Cited by the Examiner UNITED STATES PATENTS 2,768,114 10/1956Koprowski et al. 167-78 2,965,544 12/1960 Cabasso 167-78 3,080,2913/1963 Sinha et al. 167-78 3,143,470 8/1964 Wilner 16778 OTHERREFERENCES Fenje: Propagation of Rabies'Virus in Cultures of HamsterKidney Cells, Canad. Jl. Microb., vol. 6, No. 5, pp. 479-484, October1960.

Fenje: A Rabies Vaccine From Hamster Kidney Tissue Cultures: Preparationand Evaluation in Animals, Canad. Jl. Microb., vol. 6,'pp. 605-609,December 1960.

Kaplan et al.: An Indicator Plaque-Forming System for Demonstration ofInterference by Non-Cytocidal Strains of Rabies Virus, Nature, vol. 186,No. 4727, pp. 821- 822, June 4, 1960.

Kaplan et al.: Demonstration of Rabies Virus in Tissue Culture WithFluorescent Antibody Technique, Bulletin WHO, vol. 22, No. 3-4, pp.434-435, 1960.

Kissling: Growth of Rabies Virus in Non-Nervous Tissue Culture, Proc.Soc. Exp. Biol. & Med., vol. 98, No. 2, pp. 223-225, June 1958.

Nomura et al.: Growth of Rabies Virus and Development of Antigen inCulture of Chick Embryo Fragments, Japanese J1. Vet. Sci., vol. 21, No.6, pp. 112- 113, December 1959 (in Japanese), per Biological Abstracts,vol. 35, No.24, entry #71122, Dec. 15, 1960.

Ott: Preliminary Trials of a New Tissue Culture Rabies Vaccine, Vet.Med. 57 (2), pp. 158-159 (1962).

Walker et al.: The Immunizing Value of High Egg Passage Flury RabiesVirus and Its Use in Combination With the Virus of Canine Distemper,Canad. Jour. Comp. Med. & Vet. Sci., vol. 23, N0. 2, pp. 50-55, 1959.

LEWIS GOTTS, Primary Examiner.

IRVING MARCUS, Examiner.

SHEP K. ROSE, Assistant Examiner,

1. A METHOD FOR PROPAGATING A NODIFIED LIVE RABIES VIRUS WHICH COMPRISESTHE STEPS OF CULTURING CHICK EMBRYO CELLS IN VITRO N A MEDIUM CAPABLE OFFOSTERING GROWTH OF SUCH CELLS TO PRODUCE MONOLAYER TISSUE CULTURES,DECANTING THE GROWTH MEDIUM FROM THE CULTURED CELLS, WASHING SAID CELLSWITH A PROTEIN-FREE NUTRIENT MEDIUM, INOCULATING THE CULTURED CELLS WITHAN AQUEOUS SUSPENSION OF LIVE RABIES VIRUS OF THE FLURY STRAIN WHICH HASBEEN FURTHER MODIFIED BY AT LEAST ABOUT 64 SUCCESSIVE PASSAGES INEMBRYONATED EGGS, ADDING TO THE CULTURED CELLS A SYNTHETIC MAINTENANCEMEDIUM SELECTED FROM THE GROUP CONSISTING OF MEDIUM 199, MEDIUM 858 ANDEAGLE''S MEDIUM AND MAINTAINING THE RESULTING CULTURE AT A TEMPERATUREOF FROM ABOUT 34*C. TO 37*C. FOR A PERIOD OF FROM ABOUT 4 TO 14 DAYS TOACCOMPLISH MULTIPLICATION OF THE VIRUS IN SAID CELLS.